Selectins are mammalian cell-adhesion molecules specific for carbohydrate ligands. As receptors they mediate many critical cell-cell interactions. Since their discovery the academic research as well as the biotech industry has focused on developing carbohydrate based receptor blockers for the selectins identified. The best known example is 'Sialyl Lewisx, which already has entered clinical trials. However, such saccharide compounds have low affinity and, therefore, are only weakly active.
Approaches to increase affinity of the carbohydrate ligand include structural modification of the binding site and introduction of multivalency of binding sites by artificial linkages to carriers such as proteins or carbohydrates. Today it is clear that the individual selectins specifically bind different, in part mucin-like glycoproteins suggesting that the peptide backbone of these mucins contribute to the high affinity by virtue of their high content of serine and threonine as O-glycan attachment sites. Their high density of O-glycans creates a platform for multivalent presentation recently referred to as 'clustered saccharide patches'.
The present proposal aims at developing a new generation of antiinflammatory drugs by designing and producing entirely different selectin blockers which mimick the identified natural mucin ligands. We intend to set up novel production methods for the generation of glycopeptide blockers including both an industrial scale enzymatic synthesis and an industrial scale recombinant cell production using an engineered CHO cell factory.
For production of selectin blockers, two strategies will be pursued: i ) in-vivo synthesis of selectin blockers by installation of a new posttranslational biosynthetic pathway leading to the formation of branched O-glycans in CHO cells. ii) in-vitro synthesis of O-glycans using glycosyltransferases produced at industrial scale in CHO cells combined with recycling pathway for sugar-nucleotide substrates. To control the glycosylation pattern fast mapping techniques will be developed and used to study the influence of culture conditions. Besides the scale up of in-vivo and enzymatic in vi tro synthesis, aspects of downstream processing will be integrated at an early stage.
Funding SchemeCSC - Cost-sharing contracts
OX1 3QU Oxford
3584 CH Utrecht